ES2606232T3 - High performance system for CFU testing through the use of high resolution digital imaging, differential staining and automated laboratory system - Google Patents

Abstract

A method of determining the number of hematopoietic stem cells in a sample comprising: culturing hematopoietic cells obtained from a sample of a tissue or fluid containing hematopoietic cells in a medium that supports the growth of hematopoietic stem cell colonies; staining colonies in a single step with MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) without washing; take images of the colonies in the absence of a microscope; and counting erythroid colony (erythroid colony-forming unit), multiline (multiline colony-forming unit), and granulocyte-macrophage (granulocyte-macrophage colony-forming unit) precursor cells, and combinations thereof, in the sample, where the determination of the number of hematopoietic precursor cells is carried out with an image detection and quantification system comprising: a high resolution camera; appropriate light alignments; a broad spectrum flash lamp; and a positive sample identification.

Description

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DESCRIPTION

High performance system for CFU testing through the use of high resolution digital imaging, differential staining and automated laboratory system

Field of the Invention

The present invention relates to high performance systems for the objective, standardized determination of colony forming units in hematopoietic cell populations using a high resolution cell detection system and image analysis.

Background of the invention

Umbilical cord blood (CB) is an increasingly accepted source of graft for patients without related donors. Graft features currently used as determinants of graft quality and potential of CB units include enumeration of total nucleated cells (TNC), CD34 + cells and colony forming units (CFU). The number of CFUs before and after freezing / thawing of sperm is a great independent predictor of CB cell grafting. Currently, day 14 of the CFU test is the only method that determines the functional status as well as the ability to repopulate and the number of hematopoietic progenitor cells. The evaluation of CFU growth as carried out by optical microscopy and is long, subjective and difficult to standardize. In addition, current methods do not allow the counting or visualization of a sample after the test has been completed, since the sample plates are discarded at the end of test day 14.

Therefore, a high performance system to evaluate the number of CFUs, growth and identification of different types of colonies that is objective and standardized will be desirable. In addition, a system that provides positive identification of a sample and the storage of a sample image and test results for future reference will be highly desirable.

Costa GF et al Experimental Hematology 2007, 35, 1-12 describes a methodology for counting colonies of macrophage granulocyte colony forming units (CFU-GM) in vitro by data fusion.

EP 1 865 315 describes an apparatus that includes an image acquisition unit for capturing a multitude of cellular images under different image acquisition conditions together with an analysis unit for extracting a cellular position and at least one characteristic according to the crowd. of cellular images and to carry out statistical analysis with the extraneous features that serve as parameters.

Bernard R et al Phys Med Biol 2001, 46, 3061-3072 describes a method of image segmentation based on models, which employs prior knowledge about the shape of a cell colony with the aim of automatically detecting isolated, in-contact cellular colonies and overlaps of various sizes and intensities.

Compendium of the invention

According to the present invention, a method is provided for the determination of the number of hematopoietic precursor cells in a sample comprising:

culturing hematopoietic cells obtained from a sample of a tissue or fluid containing hematopoietic cells in a layer of semi-solid culture medium that supports the growth of hematopoietic precursor cell colonies;

have the colonies in a single stage with MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) without

to wash;

take pictures of the colonies in the absence of a microscope; Y

count precursor cells of erythroid colonies (erythroid colony forming unit), multilmea (multilmea colony forming unit), and granulocyte-macrophage (granulocyte-macrophage colony forming unit), and combinations thereof, in the sample,

wherein said test is carried out with an image detection and quantification system comprising:

a high resolution camera;

appropriate light alignments;

a broad-spectrum flash lamp; Y

A positive sample identification.

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The present description provides a high performance system for the objective, standardized determination of colony forming units in hematopoietic cell populations using a cellular detection system and a high resolution image analysis.

In one embodiment of the description, a standardized test of colony forming units is provided. The assay comprises the steps of obtaining a sample of a tissue or fluid containing hematopoietic cells; colonic staining; taking pictures of the colonies; colony count for each type of precursor cell; and in certain embodiments, determination of the functional status and graft potential of the colonies according to the number of precursor cells and the size of the colonies in the sample.

In yet another embodiment, a positive sample identification is provided, which comprises a sample and an identification. The sample is a cord blood unit and the identification identifies a specific cord blood unit and related information.

Brief description of the drawings

Figure 1 is an image of a 35 mm diameter well comprising CFU (colony forming units) without having (A) and having (B) after 14 days of cell culture. CFUs are identified by the following symbols: It indicates the presence of CFU-GM (granulocyte-macrophage colony forming units); 0 indicates the presence of CFU-E (erythroid colony forming units); and □ indicates the presence of CFU-GM / E (multilayered colony forming units). Positive identification is provided in the wells, and includes test date / time information, CBU sample ID, plate ID, operator identification, and extractor hood.

Detailed description of the invention

The present description provides a high performance system for the objective, standardized determination of colony forming units (CFU) in hematopoietic cell populations using colony staining and high resolution digital imaging. An informative laboratory information management system supports the testing of high performance CFUs.

In one embodiment of the description, a standardized test of colony forming units is provided. The assay comprises the steps of obtaining a sample of a tissue or fluid containing hematopoietic cells; culturing the cells in a medium that supports the growth of hematopoietic precursor cell colonies; have colonies; take pictures of the colonies; colony count for each type of precursor cell; and in certain embodiments, determine the functional status and graft potential of the colonies according to the number of precursor cells and the size of the colonies in the sample.

Hematopoietic cells can be obtained from an appropriate source, such as blood (peripheral, umbilical cord blood), bone marrow, placenta, etc. In one embodiment, the sample is umbilical cord blood. The cells obtained can be cultured in a standard medium that maintains the hematopoietic precursor cell colonies for a period of time, such as approximately 1-21 days, approximately 5-14 days, or approximately 10-14 days. In one embodiment, the cells are grown for 14 days. Examples of suitable media include, but are not limited to AMBION® (Invitrogen), STEMLINE® (Sigma), and STEMSPAN®, ALDEFLUOR®, METHOCULT®, and ALDECOUNT® (Stem Cell Technologies). In one embodiment, it is possible to selectively stimulate the proliferation of discrete cell lines using the appropriate growth factors. Examples of appropriate growth factors for the selection of particular cell lines include, but are not limited to, red blood cell erythropoietin, megakaryocyte thrombopoietin, granulocyte stimulating factor (G-CSF) for granulocytes, and macrophage granulocyte stimulating factor ( GM-CSF) for the stimulation of granulocytes and macrophages. In addition, IL-3, IL-6, stem cell factor can also be used. These growth factors can be used alone in several combinations (that is, cocktails). The estimation of the number of cells after the incubation of a certain number of cells of a given type in separate and non-separated cell samples provides an alternative method of estimating the proliferative capacity of the corresponding cell line in the sample. Thus, the acquisition of colony images of all culture plates can be extended to allow numerical estimation of cell numbers and therefore, of actual cell propagation by comparison with the number of cells sown. In certain embodiments, a subpopulation of CD34 + cells or enriched cells of light density or a shaving product may be isolated prior to the culture stage.

Images of the colonies are taken. Using the described system, CFUs can be visualized, differentiated and objectively counted and digital images can be stored for future review and / or reclassification. The system provides computerized information about optical test parameters. A combination of high resolution imaging, staining at an optional stage, and the traditional CFU test overcomes the technical challenges of conventional testing. The system supports standardization, classification, and reproducibility and high counting performance. The image detection and quantification system comprises a high resolution camera, suitable light alignments, a broad-spectrum flash lamp, and positive sample identification.

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In one embodiment, a detection system supports the testing of colony forming units. In one embodiment of the detection system, an image acquisition system is provided comprising a high-speed full-color image surface (for example, 1/125 s) of size comparable to the sample (35 mm plate diameter ). The image system has a ccd detector (for example, approximately 39 Megapixel or greater) and results in an image resolution of the order of approximately 7 microns per pixel. This is then coupled with high-distortion free 1: 1 macro focusing lenses and a stage designed as it minimizes light scattering, provides reproducible positioning between samples and allows the incorporation of human and machine-readable identification (i.e., barcode ) printed directly on each image. The excitation consists of a lamp of uniform and intense flashes of broad spectrum (approximately 1/500 s). A unique vibration-free image is then acquired through a high-performance computer for subsequent detailed image analysis, storage and retrospective studies. In one embodiment, the image system is an SLR camera manufactured by Hasselblad and the light source manufactured by Broncolor. However, any suitable camera and light source can be used. The image detection system allows the acquisition of an image of the CFU test culture plate where the CFUs grown in the culture can be clearly seen with a clear background. An additional advantage of this system is that it allows a single sample to be displayed in multiple days in exactly the same physical position due to the alignments provided in the described system. Reproducibly aligning the sample for imaging allows an entire well plate or even a single colony to be displayed repeatedly over a period of time. The growth of the colony or colonies can be traced over time by comparing the images taken on different days. For example, an image of a sample can be compared on day 1, day 5 and day 14. In this way, the growth of a colony of interest or multiple colonies of interest can be monitored.

The crop is tine. The culture is in a single stage. The cells are stained in a single stage with MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) without any washing step, which allows an even better definition of the forming units multilayered colonies (CFU-GM / E), erythroid colony forming units (CFU-E), and granulocyte-macrophage colony forming units (CFU-GM) giving a specific color for each type (dark violet, red, and light violet, respectively) against a uniformly clear background. Specific colors are generated after a time of incubation and dilution of specific MTT. For example, in one embodiment, the incubation time may be approximately 10-45 min. In another embodiment, the incubation time is approximately 15-40 min. In yet another embodiment, the incubation time is approximately 30-40 min. The dilution of the dye may vary according to the desired staining. When MTT is used, a MTT dilution of 1/2 to about 1/10 can be used. In one embodiment, a MTT dilution of 1/5 is used. In one embodiment, staining can be obtained with 30 min incubation at 37 ° C followed by 10 min at room temperature, with a MTT dilution of 1/5.

The images can be taken before and / or after staining the culture plates. In one embodiment, a culture image is taken before and after staining. In this embodiment, cultures can be easily classified and enumerated by comparison of cells and colonies not had and had if desired (See Figure 1).

In one embodiment, a computerized laboratory information system (LIMS) is used to monitor and document each stage of the CFU test system. In one embodiment, LIMS provides storage of the images of the culture plates that are linked by unique barcode ID labeling a specific CB unit. This provides positive identification of the sample linked to a specific CB unit and stores additional information such as incubator location, plating and counting dates, technician ID, as well as detailed colony information (See Figure 1).

An additional advantage of LIMS is the portability of the information stored in relation to the CB unit. That is, images and data associated with a given sample can be easily sent before, or with, the shipment of a CB unit that is requested for a transplant recipient. In this way, the recipient's doctor can see images of the sample and test, and evaluate the quality and graft potential of the CB sample there. The stored information can be sent by mail, by fax, and / or electronically. Greater portability is achieved when stored information is sent electronically.

In one embodiment, the number and functional status of the colonies can be compared with and used to estimate the graft potential of the cells in the sample (can be determined). This is achieved using standard, standard statistical methods in the analysis of weather outcomes, such as the Kaplan Meyer probability estimator (Kaplan, EL & Meier, P. (1958). “Nonparametric estimation from incomplete observations”, JOURNAL Of THE AMERICAN STATISTICAL ASSOCIATION 53: 457-481) or the cumulative incidence analysis (Gray RJ (1988) A class of K-sample tests for comparing the cumulative incidence of competing risk, ANNALS OF STATISTICS, 16: 1141-1154).

To date, the CFU test of day 14 is the only test that determines the functional status as well as the number and capacity to repopulate hematopoietic progenitor cells and is routinely carried out in laboratories. The CFU growth evaluation is carried out manually by optical phase contrast microscopy and the CFU colonies have to be classified as CFU-GM / E, CFU-E or CFU-GM based on morphology, color and size and later, counted manually. Thus, the current CFU trial is long, subjective, difficult to standardize and not practical when a large number of tests have to be tested daily.

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samples. In addition, the sample culture plates cannot be stored and therefore, all samples must be analyzed at the end of the culture (day 14) and there is no possibility of revision, discussion, counting and / or reclassification of colonies if needed before the plates are discarded.

On the contrary, the system of quantification and detection of image, assay, and methods described add the advantage of processing a large number of samplesMa. The images can be stored for future review and / or reclassification of colonies. Since the specific color that each of the colonies acquires after having, the classification is objective and the enumeration of an entire plate can be achieved quickly. More importantly, the new detection system supports positive sample identification, standardization and gives high reproducibility to the assay that can be easily implemented in laboratories where large numbers of samples need to be tested daily.

In addition, the traditional CFU assay requires the use of an inverted phase optical microscope to classify and enumerate CFUs grown on a plate, which is subjective, and long, and not practical for enumerating a large number of culture plates. Using the system described herein, there is no need for a microscope. The clear definition of the different types of colonies allows a rapid classification and enumeration of colonies and therefore helps to increase productivity. Colony images can be captured using a variety of spectroscopic techniques, such as but not limited to, absorption and fluorescence. In this way, the counting and classification of colonies between different laboratories can be standardized since it results in greater reproducibility than conventional methods of classification / enumeration. The images can be stored electronically for future review and recount by doctors. Colony enumeration can be automated using colored images. In addition, images can be easily retrieved through the barcode ID that links them to a specific sample.

A computerized procedure allows the review of the entire CFU test process (date and time of sample loading, date and time of sample plating, date and time of image sampling). The technologies that carried out each stage can be identified by labels with barcode ID in the image ensures the identification of positive sample and is linked to a specific sample. In addition, the test, method and system described allow the processing of a large number of samples / day, making this a high performance system. In one embodiment, images of approximately 100 plates can be taken and taken in approximately 2 hours. In another embodiment, images of approximately 80 plates can be taken and taken in less than approximately 2 hours. In yet another embodiment, approximately 60 plates can be taken and taken in approximately 70 minutes.

In another embodiment, a computerized counting program based on current image analysis methods for the identification of objects in the field of vision is provided herein. In one embodiment, the limits of the colony are derived and improved and differentiation is achieved. This allows a more accurate colony count and total area of each type of colony present, which can be correlated with other parameters, such as p antigen markers. eg, CD34, CD38, CD79, number of cells, demographic characteristics of the donor, and result of the transplant, drug toxicity, evaluation of new growth factors, etc. In addition, the assay, method and detection and imaging system provided herein can solve current experimental counting problems due to emerging colonies or those that have exploded in multiple colonies.

In one embodiment, the analysis can be expanded by locating the 2-dimensional boundaries of a colony in the field of vision, which allows the resolution of different colonies growing in close proximity and the calculation of the area occupied by each colony. The definition of the edge of the colony also leads, in addition, to a calculation of the total number of colonies on a plate. When coupled with discriminatory staining and / or cell markers that define cell types in the colonies, this approach can provide a direct 2- or 3d-dimensional numerical estimate (area or volume) of the ability to proliferate hematopoietic cells of cord blood and / or other samples, which is of greater precision than solely depending on the number of colonies. More specifically, in certain embodiments, obtaining estimates of the third dimension (average depth) using high resolution depth of field measurements or assuming approximately that the average depth is a function of the visible area and the thickness of the semi growth medium layer -Solid, volume estimation is allowed. The volume of a colony gives an approximate number of cells and by addition allows the estimation of the total number of cells in all colonies on a plate or specifically in all colonies of a defined cell type.

In addition, depending on the mechanical components used in the stage and optics, detection and spectroscopic resolution can detect colonies growing at different dimensional levels. In certain embodiments, the computerized alignment of images not taken and taken from an individual crop can be used for a time-dependent comparison between colonies during the day test procedure 14.

The photographic images obtained using the described test and the image detection and quantification system allow computerized storage of test results as part of a database and are useful for supporting weather applications, including the launch of cord blood units for hematopoietic transplant

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Example 1

After 14 days of CB culture (CFU test, Stem Cell Technologies), an image of a 35 mm plate was captured using a digital image system based on a high resolution camera that achieves a resolution of 7, 6 | im per pixel and also allows a clear view of all colonies on the plate with their bar code identifications. A short one-step staining protocol with MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) allows the definition of CFU-GM / E and CFU-E (dark violet / red) and CFU-GM (light violet) representing each cell type in a specific color against a uniform light background (See Figure 1). A good correlation was observed after comparing the new strategy against the traditional enumeration using the microscope (linear R2 = 0.95; n = 122 culture plates evaluated). Low variation was observed after 151 cultures were independently classified and listed by three different operators (% CV (coefficient of variation) = 8.9%; interval 1-27%) (microscope). Sample plating introduced variation in the CFU test, in an experiment where nine samples were evaluated by multiple plating (% intra-assay CV = 21.9%; range 3.4-34.5% and% CV intra -assay = 23.3%; range 12.6-35%).

In another embodiment, a computer-based laboratory information management system (LIMS) is provided to store the data related to a culture plate that is linked by a unique barcode identification (ID) labeled to a CB unit. specific. Additional information that can be linked to the CB unit through the barcode ID includes the CB image, incubator location, plating dates and counting, as well as detailed colony enumeration. This system has been tested in more than 8,000 units of CB in duplicate. The specific coloration of the CFU colonies allows a faster classification and enumeration and thus allows a more precise analysis of CFU colonies and allows a better determination of their relationships with antigen surface markers such as CD34 + cell content, drug toxicity and transplant grafting.

Unless otherwise indicated, all numbers that express quantities of ingredients, properties such as molecular weight, reaction conditions, and others used in the requirements and claims should be understood as modified in all cases by the term "approximately." Therefore, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending on the desired properties that are intended to be obtained by the present invention. At least, and not as an attempt to limit the application of the equivalent fee to the scope of the claims, each numerical parameter should be constructed at least in the light of the number of significant digits reported and applying ordinary rounding techniques. Although the numerical intervals and parameters exposed to the wide scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective test measurements.

The terms "a", "a", "the" and similar references used in the context of the description of the invention (especially in the context of the following claims) should be constructed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. The enumeration of ranges of values in this memory is intended to serve as a shorthand reference method for each separate value that falls within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were listed individually in this memory. All methods described herein may be carried out in any suitable order unless otherwise indicated herein or clearly otherwise contradicted by the context. The use of any and all examples, or illustrative language (eg, "as") provided herein is merely intended to better illuminate the invention and does not represent a limitation on the scope of the invention claimed otherwise. No language should be constructed in the specification as indicating any essential element not claimed for the practice of the invention.

Specific embodiments described herein may also be limited in the claims using the language "consisting of" or "consisting essentially of". When used in the claims, whether presented or added for amendment, the term "which consists of" excludes any element, step, or ingredient not specified in the claims. The term "essentially consisting of" transitions limits the scope of a claim to the specified materials or stages and those that do not materially affect the basic feature (s).

In addition, numerous references to patents and printed publications have been made throughout this specification.

Claims (6)

5 10 fifteen twenty 25 1. A method for determining the number of hematopoietic precursor cells in a sample comprising: culturing hematopoietic cells obtained from a sample of a tissue or fluid containing hematopoietic cells in a medium that supports the growth of hematopoietic precursor cell colonies; having the colonies in a single stage with MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) without washing; take pictures of the colonies in the absence of a microscope; Y count precursor cells of erythroid colonies (erythroid colony forming unit), multilmea (multilmea colony forming unit), and granulocyte-macrophage (granulocyte-macrophage colony forming unit), and combinations thereof, in the sample, where the determination of the number of hematopoietic precursor cells is carried out with an image detection and quantification system comprising: a high resolution camera; appropriate light alignments; a broad-spectrum flash lamp; Y A positive sample identification. 2. The method according to claim 1, further comprising isolating one or more selected from the group consisting of a subpopulation of CD34 + cells, enriched cells of light density, a product of aferesis, and a combination thereof, before the stage of cultivation 3. The method according to claim 1, wherein the different types of precursor cells are stained in different colors. 4. The method of claim 1, further comprising a positive sample identification comprising the sample and an identification, wherein the identification identifies a specific sample and information related thereto. 5. The method of claim 1, wherein the sample is identified with an identification tag that identifies a specific sample and information related thereto. 6. The method of claim 1, further comprising the step of taking pictures of the colonies before having.